Collagen gels, bandages, sponges and fibers have been used as carrier for stem cells. Collagen is a natural protein present in many tissues. It supplies tensile strength to the tissues as well as providing attachment sites for cells to bind, migrate and proliferate. However, collagen gels and pulverized collagen lack tensile strength and tend to dissipate in the tissue under pressure and thus are not well suited to the task of maintaining cells in defined locations.
Collagen can be isolated in pure form from a variety of tissues as a soluble protein which can be reconstituted into fibers. In general, fibers lack the superstructure they exhibit in different tissues. This has limited the success of using collagen in many medical applications.
Tissue damage to heart, striated muscle, skin, bone and cartilage, spinal cord, etc. often progresses and causes the breakdown of normal surrounding tissue increasing the area damaged and seriously impairing tissue function. Current concepts suggest that various factors or cells could be introduced into the damaged area by direct injection and prevent further damage and even restore the damaged area. Indeed, preclinical as well as clinical trials have shown improved heart function when mesenchymal stem cells are injected into the site of tissue damage. However, such studies have also shown that significant amounts of the injected cells leak out of the tissue and most of the cells remaining rapidly die. Thus there is a need for devices which allows delivery of various types of cells to specific sites in damaged tissue and maintain their survival and expansion. However, the physical properties of different tissues vary strikingly (compare heart muscle with striated muscle with skin or spinal cord. Thus, delivery vehicles should have sufficient strength to allow them to be placed in a specific tissue without causing alterations in tissue function due to poorly matched physical factors. This calls for materials whose physical properties are suitable for the tissue in which they are to be used. Also, cells require factors and surfaces to support their survival, migration and production of specific repair factors. In brief, the treatment of damaged tissues should be improved by novel devices which would deliver and maintain stem and other cells in specific sites to allow them to survive and produce factors that sustain tissue at risk.
Tendon and ligament injuries are among the most common health problems affecting the adult population. It is estimated that 175,000 Anterior Cruciate Ligament (ACL) reconstructions were performed in the year 2000 in the United States at a cost of more than $2 billion. There are 50,000 patients requiring surgical repair of rotator cuff and 5 million new cases of tennis elbow each year in the United States alone. In addition, 11% of regular runners suffer from Achilles tendinopathy. In 2000, treatments for shoulder pain cost the United States government up to $7 billion and the total cost for tendon and ligament injury has been estimated at $30 billion annually; see Chen, J., Xu J., Wang A., Zheng M., 2009. Scaffolds for tendon and ligament repair: review of the efficacy of commercial products. Expert Rev. Med. Devices. 6. 1:61-73.
Current methods to repair tendon and ligaments of the hand and foot have had only limited success. In some cases, the joints are immobilized for four weeks while scar tissue builds up. This scar tissue, which has poor mechanical properties, must assume the function of the original tendon/ligament but has a high rate of failure. In other cases non-absorbable sutures anchored to bone are used. These sutures lack the appropriate elasticity and often evoke an immune response. In other cases, repair is not possible and the replacement procedures available have various limitations.
Current clinical and research practice utilizes animal and human grafts as the primary material in daily clinical practice to repair tendon and ligament defects. Biological substitutes include autografts, allografts, and xenografts.
Autologous grafts of patellar tendon and hamstring tendons are considered the “gold standard” in tissue repair and are usually preferred to avoid rejection. However, these autografts suffer from a number of disadvantages. For example, autografts require additional surgery on the patient which may cause donor site morbidity, increased recovery time, and/or possible pain at the harvesting site plus harvest site infection, nerve injury, and patellar fracture. An additional disadvantage in the use of the hamstring tendon for ACL reconstruction is the lack of efficient tendon-bone healing, which affects patient recovery due to instability of the tendon-bone interface.
Allografts are obtained from tendons, dermis and other tissues of cadavers. Xenografts are harvested from animal tendons, small intestine submucosa, dermis and skin, and pericardium. Allografts and xenografts are primarily composed of type I collagen with similar mechanical properties to human tendons. However, allo- and xenografts can potentially transmit disease or infection, and may elicit an unfavorable immunogenic response from the host.
Synthetic grafts exhibit excellent short-term results but the long-term clinical outcome is poor, with a failure rate of 40 to 78% from fragmentation, stress shielding of new tissue, fatigue, creep, and wear debris, which can eventually lead to arthritis and synovitis.
Current cell-seeding techniques include: a) delivering cell-gel composites into the region of the scaffold and b) delivering cell suspensions into scaffolds in a static or dynamic situation. However, there are some disadvantages to these techniques, such as the low efficiency of cell attachment to dense fibrous matrix or scaffolds and the weak mechanical strength of gel systems. These disadvantages make it very difficult to seed a significant number of cells on dense tissue grafts. Thus the limitations of current technology prohibit the use of stem cells to improve the efficiency of large tissue grafts for tissue repair. To address these limitations partial thickness incisions and ultrasonication were developed to allow the seeded cells to infiltrate the tendon in culture prior to implantation. Without incisions or ultrasonication, extrinsic cells seeded onto the tendon surface have difficulty penetrating the tendon. However, with incision and ultrasonication, the mechanical strength of the grafts is reduced which limits the potential of clinical application.
Several research groups have attempted to tissue engineer neo-ligaments in vitro for tendon and ligament replacements as described in Hairfield-Stein M. et al. 2007. Development of Self-Assembled, Tissue-Engineered Ligament from Bone Marrow Stromal Cells. Tissue Engineering. 13. 4:703-710. One of the first promising results in this direction has been published by Goldstein J D et al. in Goldstein, J D, et al. 1989. Development of a Reconstituted Collagen Tendon Prosthesis. The Journal of Bone and Joins Surgery. 71. A. 8: 1183-1191; and Dunn et al. Dunn, M G. et al. 1992. Anterior cruciate ligament reconstruction using a composite collagenous prosthesis. A biomechanical & histological rabbit study. Am J Sports Med. 20. 507-515. Constructed scaffolds of collagen fibers are bundled in different configurations with and without a collagen gel to hold them together. Dunn et al. and other groups used collagen scaffolds with and without seeded cells. The main problems of this approach have been: a) the rate of degradation of scaffold collagen was higher than the rate of newly synthesized collagen causing the neo-ligament to fail to support in vivo loading; and b) the cost of production was too high and reproducibility was low.
To reduce the rate of degradation, several research groups have used silk and other synthetic materials. This solved the enzymatic degradation problem but generated other problems including immune reactions, fibrosis, and wear debris. Attempts to eliminate extracellular matrix scaffolds have led to a new approach of so-called “functional tissue-engineering” or “spontaneous 3-dimensional tissue development” as described in Calve, S., Dennis R G, Kosnik P E, Baar K, Grosh K, Arruda E M. 2004. Engineering of functional tendon. Tissue Engineering. 10, 755-761. This method does not rely as heavily upon the material properties of pre-existing artificial or biological scaffolds which limit the physiologic or mechanical properties of the newly formed tissue. Rather media like collagen gels, fibrin gels, Matrigel, or synthetic gels are employed to orchestrate the formation of the newly formed tissue. Chen et al in Chen X, Zou X H, Yin G L, Ouyang H W. 2009. Tendon tissue engineering with mesenchymal stem cells and biografts: an option for large tendon defects? Frontiers in Bioscience. S1. 23-32 present a comprehensive review of this and other stem cell tissue engineering methods for tendon repair. However, the problems (a) and (b) highlighted above are still valid in the case of functional tissue engineering.
Several efforts have been made to render reconstituted collagen structures with some degree of orientational anisotropy. The alignment of molecules and fibrils by mechanical loading, microfluidic channels, flow and magnetic field induced alignment, electrochemical processing, interstitial flow, high magnetic field, oriented electrospinning, Langmuir-Blodgett deposition, and extrusion processes have been demonstrated in the research lab. The collagen matrices produced by each of these processes have successfully caused the alignment of different cells. However, they do not mimic natural extracellular matrices (ECMs) and lack the essential natural spatial complexity, for example, controlled fibril diameters, aligned fibrils, crimps, periodicity and angular distribution. The lack of secondary structure results in poor strength in the synthetic scaffold and affects cellular survival and behavior. Builles et al. (2007) demonstrate the importance of the cell environment on a newly synthesized ECM. Some have achieved better control of fibril size and demonstrate the formation of tissue-like patterns. However, there are problems with the size of the construct they produce and the lengthy process required. The importance of nano- and micro-structures and their orientation is especially important for tendons and ligaments, since these tissues require strength, elasticity and a guide for cells to attach and migrate for repair to occur. For example, a recent review devoted to cellular-extracellular architecture of human tissue has no discussion about fibril orientation or alignment in ECM, although it states that “the importance of scaffold architecture in tissue engineering is increasingly being realized, which has resulted in a change in trend in the designs of scaffolds, from isotropic scaffolds to heterogeneous and anisotropic “biomimetic” scaffolds, with the goal being to mimic the organization of the cells (such as alignment or clustering) and/or the ECM of the tissue under consideration.” See 30. Singh M., Tech B, Berkland C, and Detamore M S. 2008. Strategies and Applications for Incorporating Physical and Chemical Signal Gradients in Tissue Engineering. Tissue Engineering. Part B. 14. 4: 341-366.
More recently, methods to deposit collagen in ordered arrays on glass or plastic substrate to make a thin film with skin-like, tendon-like, aligned-braided, and other fibrillar structures have been described in United States Patent Application Publication No. 2009/0069893 and U.S. patent application Ser. No. 11/951,324, the entire disclosures of which are hereby incorporated by reference. Importantly, these aligned and ordered collagen deposits direct the orientation of cells applied to them. Specifically, cells attach and align on the films and migrate along the axis of alignment. Such films can be treated with factors, such as growth factors which enhance cell survival, migration and proliferation. Other biopolymers can and have been devised to deliver cells to tissues but many lack biocompatibilities, are not lost or incorporated into the tissue or have physical properties quite different from the tissue and alter its function.
Despite substantial efforts to develop biomaterials to mimic tendon, skin and other collagen-based fibrillar tissues, to date there is no industrial manufacturing method for producing these materials. Accordingly, significant further developments are much needed.